N-type and P/Q-type Ca channels are responsible for the Ca entry that initiates neurotransmitter release at most conventional fast synapses in the brain and peripheral nervous system. Ca entering through activated presynaptic Ca channels forms a local domain of high Ca concentration which activates exocytosis in the near vicinity of the Ca channel. Therefore, synaptic vesicles must dock near presynaptic Ca channels to be efficiently released. Neurotransmitter release is dependent on the fourth power of the Ca current, so small changes have large effects on synaptic transmission. Many neurotransmitters which act through G protein-coupled receptors can inhibit the activity of presynaptic Ca channels and thereby inhibit synaptic transmission. Their inhibition is relieved by strong depolarization or by phosphorylation by protein kinase C. Our results in the present project period have given important new insights into the function of presynaptic Ca channels. First, we have shown that regulation of regulation of the Ca channels by G protein-coupled receptors involves direct binding of the betagamma subunit of the G proteins to a site in the intracellular loop connecting domains I and II of the alpha1 subunit. Second, we have discovered that the SNARE proteins involved in docking and exocytosis of synaptic vesicles bind to N-type and P/Q-type Ca channels via a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their alpha1 subunits. This interaction is required for efficient neurotransmission. In the next project period, we plan to build on these two advances to further define the molecular mechanism of Ca channel regulation by G proteins and to probe the molecular mechanism and functional significance of Ca channel interaction with SNARE proteins. Our Specific Aims are as follows. We will define the molecular basis for the modulation of presynaptic Ca channels by G protein betagamma subunits using a combination of in vitro binding, in vivo binding, site-directed mutagenesis, and patch clamp recording methods. We will analyze the structure and function of the synprint site of presynaptic Ca channels using in vitro binding methods together with site-directed mutagenesis to map the sites of interaction of SNARE proteins at the level of resolution of single amino acid residues. In addition, we propose to express the minimal synprint site and analyze its structure by multi-dimensional NMR. The molecular mechanism of modulation of SNARE protein binding to the synprint site by protein phosphorylation will be probed by molecular mapping of the phosphorylation sites and determination of their functional effects on binding of SNARE proteins. The functional significance of SNARE protein interaction with the synprint site for Ca channel regulation will be defined in transfected mammalian cells and in neurons. Finally, we will determine the significance of SNARE protein interaction with the synprint sites of presynaptic Ca channels for synaptic function using cultured mammalian neurons and analysis by electrophysiological and dye-release recording methods. These studies will provide novel insights into the function and regulation of presynaptic Ca channels in neurotransmitter release and will be essential to understanding the molecular basis for synaptic transmission and its modulation.